The invention relates to non-peptidic xcex1-ketoamide compounds and molecular arrays of potential protease inhibitors and the uses thereof.
Proteolytic enzymes, or proteases, are proteins that catalyze the degradation of peptide bonds in protein and peptide substrates. Proteases are typically categorized into four major classes (i.e., serine, aspartyl, metallo, and cysteine), classified according to the catalytic site chemical group that facilitates peptide bond hydrolysis. Proteases are involved in a wide variety of physiological and pathological processes including blood coagulation, protein turnover, complement activation, hormone processing, and cancer cell invasion.
Cysteine proteases, for example, are utilized by living organisms to perform a variety of key cellular functions, and thus are potential targets for drug discovery. For example, cathepsin B has been studied for its role in the progression of normal tissue to cancerous tissue, and the protease cruzain is believed to be essential for the parasitic infection in Chagas"" disease (a major public health problem in South and Central America, affecting about 25% of the population of those regions).
The interaction of a protease with a substrate is a highly specific binding event that is driven by, for example, favorable molecular shape recognition (i.e., between the protease and the substrate) and electrostatic e.g., charge-charge, dipolar, or van der Waals) interactions that occur upon binding. Recognition and binding typically involves 3 to 4 amino acid residues of the substrate on either side of an enzyme""s catalytic site. Although the kinetics of all proteolytic events are not fully understood, most protease-mediated catalysis occurs because the catalytic site stabilizes a transition-state, structural intermediate in the pathway to peptide-bond cleavage.
Inhibitors of proteolytic activity typically interact with a protease at its active site, preventing interaction (e.g., recognition, binding, or reaction) of enzyme and substrate. However, inhibition via allosteric change (i.e., conformational or other structural change) and co-factor binding inhibition are some other possible modes of inhibition. Potent and specific synthetic inhibitors can: 1) interact with the enzyme""s binding pocket with high affinity, and 2) interact with the catalytic site to mimic the transition state structure.
Modulation, e.g., inhibition or enhancement, of protease activity can profoundly influence biological systems, and, therefore, proteases are often chosen as targets for drug discovery. In the design of protease inhibitors, researchers have generally identified chemical structures that interact with the catalytic chemical group at the active site of the protease and find structures that mimic the transition state of the catalytic reaction. These identified structures are then linked to a di- or tri-peptide sequence that specifically binds to the active site substrate binding pockets. Peptide-based inhibitors, mimicking the primary sequence of the natural substrate, often show very high potency against the target; however, orally administered peptides generally exhibit poor bioavailability due to hydrolysis by nonspecific proteolytic enzymes in the digestive system. Substitution of the peptide portion of protease inhibitors with small organic molecules that mimic the molecular shape and charge. interactions of the peptides frequently results in improved bioavailability and oral absorption for that inhibitor.
The invention is based on new methods for making and using compounds and arrays of novel xcex1-ketoamides, and the arrays and compounds made by these methods. These novel compounds are potential inhibitors of proteolytic enzymes, particularly cysteine proteases such as cruzain. Application of the new methods has led-to the identification of a number of new inhibitors, from amongst an array of about 38,000 xcex1-ketoamide derivatives, having specific activity against two cysteine proteases: cruzain and cathepsin B. These compounds and other compounds identified by the methods described herein can be useful, for example, in developing pharmaceutical agents for the treatment of diseases (e.g., Chagas"" disease) associated with these proteases. Although the disclosed compounds have specific activity for cruzain and cathepsin B, the methods described herein can also be used to identify inhibitors of other proteases.
In one embodiment, the invention features a method for preparing a monoacylated diamine compound. The method includes the step of reacting a diamine with an xcex1-ketoester compound, under conditions such that a monoacylated diamine is prepared. The diamine can be represented, for example, by the structure Bxe2x80x94NHxe2x80x94Yxe2x80x94NHxe2x80x94C, where Y is a linker moiety (i.e., a divalent alkyl, carbocyclic, or aryl groups), and B and C can independently be hydrogen, an alkyl group, a carbocyclic group, or an aryl group. The xcex1-ketoester can be represented, for example, by the structure: 
where A and R2 can independently be an alkyl group, a carbocyclic group, or an aryl groups.
A functionalized xcex1-ketoamide compound can also be formed, for instance, by preparing a monoacylated diamine by the above method, then reacting the monoacylated diamine with an electrophile (e.g., an alkoxymethylene oxazolone, an acid halide, an isocyanate, an isothiocyanate, an anhydride, a halotriazine, a Michael acceptor, an aldehyde, or a ketone).
In another embodiment, the invention features a method for preparing a plurality (e.g., 100 or more, or 1000 or more) of xcex1-ketoamide compounds. The method includes the step of reacting a plurality of diamine compounds with a plurality of xcex1-ketoester compounds, under conditions whereby a plurality of xcex1-ketoamide compounds is prepared.
The plurality of diamines can include, for example, a diamine that can be represented by the structure Bxe2x80x94NHxe2x80x94Yxe2x80x94NHxe2x80x94C, where Y, B, and C are defined as above. Each of the xcex1-ketoester compounds can be, for example, represented by the structure: 
wherein A and R2 are defined as above.
The method can also include the step of reacting the plurality of xcex1-ketoamide compounds with a plurality of electrophiles (alkoxymethylene oxazolones, acid halides, isocyanates, isothiocyanates, anhydrides, halotriazines, Michael acceptors, aldehydes, ketones, or combinations thereof), such that a plurality of functionalized xcex1-ketoamide compounds is prepared.
The plurality of compounds can, for example, be arranged in a spatially addressable array format. An array produced by this method is contemplated.
Still another embodiment of the invention features an array of xcex1-ketoamide compounds. Each of the xcex1-ketoamide compounds can be represented, for example, by the formula: 
where A can be an alkyl group, a carbocyclic group, or an aryl group; B and C independently can be hydrogen, an alkyl group, a carbocyclic group, or an aryl group; Y can be a divalent alkyl group, carbocyclic group, or aryl group; and D can be hydrogen, an alkyl group, a carbocyclic group, an aryl group, or xe2x80x94C(X)xe2x80x94Zxe2x80x94W, where X is O or S, Z is a single bond or NR; and R and W independently can be hydrogen, an alkyl group, a carbocyclic group, or an aryl group.
The array can include, for example, at least about 100 compounds, or at least about 1000 compounds.
D can be, for example, the triazolinyl moiety: 
where X and Z independently can be an unsubstituted, monosubstituted, or disubstituted amino group, a thioalkyl group, a thioaryl group, an alkoxy group, an aryloxy group, a halogen (e.g., F, Cl, Br, or I), an alkyl group, a carbocyclic group, or an aryl group.
Alternatively, D can be the oxazolinyl moiety: 
where X can be an alkyl group, a carbocyclic group, or an aryl group.
A method of identifying a compound having a specific characteristic (e.g., inhibition of an enzyme, or other biological activity or interaction) is also contemplated. The method includes screening any of the arrays described above with an assay capable of detecting the presence of a compound having the specific characteristic. For example, compounds that inhibit proteases (e.g., a serine protease such as xcex1-thrombin, Factor Xa, plasmin, or trypsin; a cysteine protease such as cruzain, cathepsin B, cathepsin K, papain, or calpain; a metalloprotease such as TACE or 92 kDa gelatinase; or an aspartyl protease) can be identified.
Yet another embodiment of the invention features a non-peptidyl compound represented by the formula: 
where A-D and Y are as defined above. In particular, the compound can have, for example, one of the following six structures: 
The compound can be, for example, an inhibitor of a cysteine protease such as cruzain.
Alternatively, D can represent the triazolinyl moiety: 
wherein X and Z are as defined above for triazolinyl moieties; or the oxazolinyl moiety: 
wherein X is defined as above for oxazolinyl moieties.
In some cases, A can be either 2,4-difluorophenyl or 4-nitrophenyl. Y can be a C2-C6 alkylene. D can be hydrogen, and B and C can be identical.
For example, A can be 3-trifluoromethylphenyl, phenyl, 4-bromophenyl, 2,4-difluorophenyl, 4-nitrophenyl, 4-tert-butylphenyl, 3-tolyl, 3-methoxyphenyl, 3-fluorophenyl, or 4-methoxyphenyl; D can be 2-thienyl, 2-naphthyl, p-biphenyl, m-tolyl, 4-trifluoromethylphenyl, 2-furyl, 2-chlorophenyl, o-tolyl, 4-t-butylphenyl, 3-methoxyphenyl, 2,4-dichlorophenyl, 3-nitrophenyl, 4-bromophenyl, 1-naphthyl, 3-furyl, 3,4-methylenedioxyphenyl, 3-pyridyl, p-tolyl, 4-chlorophenyl, or 4-nitrophenyl; and the combination of Y, B, and C can be selected from one of the following combinations: Y is ethyl and B and C are methyl; Y is propyl and B and C are methyl; Y is hexyl and B and C are methyl; Y, B, C, and the two nitrogen atoms form a piperazine; Y, B, C, and the two nitrogens form a homopiperazine; Y is butyl and B and C are hydrogen; Y is cyclohexyl attached to the nitrogens at the 1 and 3 positions and B and C are methyl; or Y is propyl and B and C are hydrogen. Y can be ethylbenzene-2xe2x80x2,4-diyl or toluene 1xe2x80x2,4-diyl.
In some cases, Y can be ethylbenzene-2xe2x80x2,4-diyl or toluene 1xe2x80x2,4-diyl. A can be 2-benzo[b] thienyl. B and C can both be hydrogen.
A composition comprising any of the above compounds together with an acceptable (i.e., pharmacologically safe) excipient in also contemplated, as is a method of treating a subject suffering from Chagas"" disease by administering to the subject an effective protease-inhibiting amount of any of the above compositions.
Another embodiment of the invention features a method for inhibiting a protease (e.g., a serine protease, or a cysteine protease such as cruzain). The method includes the steps of contacting the protease with a compound represented by the formula: 
where A-D and Y are defined as above.
In a specific example, the compound can have one of the following six structures: 
In another embodiment, the invention features a method for making an xcex1-ketoamide aminomethylene oxazolone. The method includes the steps of reacting a diamine of formula: 
with an xcex1-ketoester of formula: 
to form an xcex1-ketoamide; and then reacting the xcex1-ketoamide with an alkoxymethylene oxazolone of formula: 
where A-D and Y are as defined above.
For example, the diamine can be ortho-aminobenzoylamine, meta-aminobenzoylamine, para-aminobenzoylamine, N,Nxe2x80x2-dimethyl-1,2-ethylenediamine, N,Nxe2x80x2-dimethyl-1,3-propanediamine, N,Nxe2x80x2-dimethyl-1,6-hexanediamine, piperazine, homopiperazine, 1,4-diaminobutane, 1,3-cyclohexanebis(methylamine), or 1,3-diaminopropane; the xcex1-ketoester can be alkyl 3-trifluoromethylbenzoylformate, alkyl benzoylformate, alkyl 4-bromobenzoylformate, alkyl 2,4-difluorobenzoylformate, alkyl 4-nitrobenzoylformate, alkyl 4-tert-butylbenzoylformate, alkyl 3-methylbenzoylformate, alkyl 3-methoxybenzoylformate, alkyl 3-fluorobenzoylformate, or alkyl 4-methoxybenzoylformate (where alkyl is methyl, ethyl, or other unhindered or activated aliphatic substituent to form an xcex1-ketoamide); and the alkoxymethylene oxazolone can be 2-thienyl alkoxymethylene oxazolone, 2-naphthyl alkoxymethylene oxazolone, p-biphenyl alkoxymethylene oxazolone, m-tolyl alkoxymethylene oxazolone, 4-trifluoromethylphenyl alkoxymethylene oxazolone, 2-furyl alkoxymethylene oxazolone, 2-chlorophenyl alkoxymethylene oxazolone, o-tolyl alkoxymethylene oxazolone, 4-t-butylphenyl alkoxymethylene oxazolone, 3-methoxyphenyl alkoxymethylene oxazolone, 2,4-dichlorophenyl alkoxymethylene oxazolone, 3-nitrophenyl alkoxymethylene oxazolone, 4-bromophenyl alkoxymethylene oxazolone, 1-naphthyl alkoxymethylene oxazolone, 3-furyl alkoxymethylene oxazolone, 3,4-methylenedioxyphenyl alkoxymethylene oxazolone, 3-pyridyl alkoxymethylene oxazolone, p-tolyl alkoxymethylene oxazolone, 4-chlorophenyl alkoxymethylene oxazolone, or 4-nitrophenyl.
Yet another embodiment of the invention is a method for making an xcex1-ketoamide thiourea. The method includes the steps of reacting a diamine of formula: 
with an xcex1-ketoester of formula: 
to form an xcex1-ketoamide; and reacting the xcex1-ketoamide with an isothiocyanate of formula: 
where A-D and Y are defined as above.
An array of compounds is also contemplated, where each of the compounds in the array is prepared according to one of the above methods.
xe2x80x9cStructural diversity elementsxe2x80x9d are chemical functional groups and can include linear chain or branched chain alkyl groups, carbocyclic groups, aryl groups, and heteroatomic functional groups such as nitro groups, sulfonyl groups, and other nitrogen, oxygen, sulfur, or halogen bearing groups. In an array that includes a plurality of compounds, structural diversity elements are the variable parts of the compound. In contrast, the xe2x80x9cmolecular core structurexe2x80x9d is invariant, common to each compound in the array.
The terms xe2x80x9cbonded,xe2x80x9d xe2x80x9cbinding,xe2x80x9d xe2x80x9cbinds,xe2x80x9d or xe2x80x9cbound,xe2x80x9d as used herein, can refer to, for example, covalent, ionic, van der Waals, or hydrophobic interactions. Coordination complexes and hydrogen bonding are also contemplated. Typically, the bonding interactions are reversible, but can be irreversible in some cases.
xe2x80x9cAlkyl groupsxe2x80x9d should be construed to include both linear chain and branched chain derivatives of any substituted or unsubstituted acyclic carbon-containing moieties, including alkanes, alkenes, and alkynes. Alkyl groups having one to five, ten, twenty, or even more carbon atoms are possible. Examples of alkyl groups include lower alkyls, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl; higher alkyls, for example, octyl, nonyl, and decyl; lower alkenyls, for example, ethenyl, propenyl, propadienyl, butenyl, butadienyl; higher alkenyls such as 1-decenyl, 1-nonenyl, 2,6-dimethyl-5-octenyl, and 6-ethyl-5-octenyl; and alkynyls such as 1-ethynyl, 2-butynyl, and 1-pentynyl. Other linear and branched alkyl groups are also within the scope of the present invention.
In addition, such alkyl groups can also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Functional groups include, but are not limited to, tertiary amine, amide, ester, ether, and halogen, i.e., fluorine, chlorine, bromine and iodine. Specific substituted alkyl groups can be, for example, alkoxy such as methoxy, ethoxy, butoxy, and pentoxy; dimethylamino, diethylamino, cyclopentylmethylamino, benzylmethylamino, and dibenzylamino; formamido, acetamido, or butyramido; methoxycarbonyl or ethoxycarbonyl; or dimethyl or diethyl ether groups.
xe2x80x9cCarbocyclic groupsxe2x80x9d include both substituted and unsubstituted, cyclic, carbon-containing moieties such as cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl. Such cyclic groups can also contain various substituents in which one or more hydrogen atoms have been replaced by a functional group. Such functional groups include those described above, as well as lower alkyl groups as described above. The cyclic groups of the invention can also include one or more heteroatoms, for example, to form heterocyclyls.
xe2x80x9cAryl groupsxe2x80x9d include substituted and unsubstituted hydrocarbon rings bearing a system of conjugated double bonds, usually comprising (4n+2) pi bond electrons, where n is an integer equal to or greater than 0. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, tolyl, xylyl and the like. Aryl groups can also include aryloxy, aralkyl, aralkyloxy and heteroaryl groups, e.g., pyrimidine, morpholine, piperazine, piperidine, benzoic acid, toluene, thiophene, and the like. These aryl groups can also be substituted with any number of a variety of functional groups. In addition to the functional groups described above in connection with substituted alkyl groups and carbocyclic groups, functional groups on the aryl groups can also include other nitrogen, oxygen, sulfur, or halogen bearing groups.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
An advantage of the new xcex1-ketoamide-based inhibitors is that they are non-peptidyl. Low molecular weight organic molecules are often superior to peptidyl compounds as protease inhibitors, primarily due to such factors as their stability in the acidic environment of the digestive system, good transport into the vascular system, and high bioavailability to the target tissue. The new compounds and arrays disclosed herein can lead to the identification of new classes of potentially reversible protease inhibitors that are both potent and specific. For example, potent in vitro inhibitors of the cysteine protease cruzain are disclosed herein.
In addition, the xcex1-ketoamide-based inhibitors appear to demonstrate reversible binding kinetics. The pharmacokinetics (i.e., tissue-residence time, renal clearance) of reversible inhibitors are generally superior to those of irreversible inhibitors in target tissue. For example, these properties allow a physician to more easily maintain therapeutic doses of the drugs in the patient""s serum. Thus, the new xcex1-ketoamide-based inhibitors are potentially superior to the known cysteine protease inhibitors (e.g., chloromethylketones, epoxy-succinyl compounds, and vinyl sulfones), all of which bind irreversibly.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.